ecallantide and Inflammation

Topics: Animals; Atherosclerosis; Blood Coagulation; Bradykinin; Cell Movement; Cell Proliferation; Complement Pathway, Classical; Factor XI; Factor XII; Fibrinolysis; Fibroblasts; Humans; Inflammation; Kininogen, High-Molecular-Weight; Mice; Neutrophils; Plasma Kallikrein; Prekallikrein; Wound Healing

The contact activation system (CAS) and kallikrein/kinin system (KKS) are older recognized biochemical pathways that include several proteins that skirt the fringes of the blood coagulation, fibrinolytic, complement and renin-angiotensin fields. These proteins initially were proposed as part of the hemostatic pathways because their deficiencies are associated with prolonged clinical assays. However, the absence of bleeding states with deficiencies of factor XII (FXII), prekallikrein (PK) and high-molecular-weight kininogen indicates that the CAS and KKS do not contribute to hemostasis. Since the discovery of the Hageman factor 60 years ago much has been learned about the biochemistry, cell biology and animal physiology of these proteins. The CAS is a pathophysiologic surface defense mechanism against foreign proteins, organisms and artificial materials. The KKS is an inflammatory response mechanism. Targeting their activation through FXIIa or plasma kallikrein inhibition when blood interacts with the artificial surfaces of modern interventional medicine or in acute attacks of hereditary angioedema restores vascular homeostasis. FXII/FXIIa and products that arise with PK deficiency also offer novel ways to reduce arterial and venous thrombosis without an effect on hemostasis. In summary, there is revived interest in the CAS and KKS due to better understanding of their activities. The new appreciation of these systems will lead to several new therapies for a variety of medical disorders.

Topics: Animals; Blood Coagulation; Blood Coagulation Disorders; Bradykinin; Factor XII; Factor XIIa; Hemostasis; Homeostasis; Humans; Inflammation; Kallikrein-Kinin System; Kininogen, High-Molecular-Weight; Mice; Plasma Kallikrein; Prekallikrein; Receptors, Bradykinin; Thrombosis

The kallikrein kinin system (KKS) consists of serine proteases involved in the production of peptides called kinins, principally bradykinin and Lys-bradykinin (kallidin). The KKS contributes to a variety of physiological processes including inflammation, blood pressure control and coagulation. Here we review the protein structural data available for these serine proteases and examine the molecular mechanisms of zymogen activation and substrate recognition focusing on plasma kallikrein (PK) and tissue kallikrein (KLK1) cleavage of kininogens. PK circulates as a zymogen bound to high-molecular-weight kininogen (HK). PK is activated by coagulation factor XIIa and then cleaves HK to generate bradykinin and factor XII to generate further XIIa.A structure has been described for the activated PK protease domain in complex with the inhibitor benzamidine. Kallikrein-related peptidases (KLKs) have a distinct domain structure and exist as a family of 15 genes which are differentially expressed in many tissues and the central nervous system.They cleave a wide variety of substrates including low-molecular-weight kininogen (LK) and matrix proteins. Crystal structures are available for KLK1, 3, 4, 5, 6 and 7 activated protease domains typically in complex with S1 pocket inhibitors. A substrate mimetic complex is described for KLK3 which provides insight into substrate recognition. A zymogen crystal structure determined for KLK6 reveals a closed S1 pocket and a novel mechanism of zymogen activation. Overall these structures have proved highly informative in understanding the molecular mechanisms of the KKS and provide templates to design inhibitors for treatment of a variety of diseases.

Topics: Amino Acid Sequence; Animals; Blood Pressure; Catalysis; Catalytic Domain; Enzyme Precursors; Factor XIIa; Humans; Inflammation; Kallikrein-Kinin System; Kininogens; Models, Molecular; Molecular Sequence Data; Peptide Hydrolases; Plasma Kallikrein; Sequence Homology, Amino Acid; Serine Proteases; Substrate Specificity; Tissue Kallikreins

Plasma kallikrein (PK) is a serine protease generated from plasma prekallikrein, an abundant circulating zymogen expressed by the Klkb1 gene. The physiological actions of PK have been primarily attributed to its production of bradykinin and activation of coagulation factor XII, which promotes inflammation and the intrinsic coagulation pathway. Recent genetic, molecular, and pharmacological studies of PK have provided further insight into its role in physiology and disease. Genetic analyses have revealed common Klkb1 variants that are association with blood metabolite levels, hypertension, and coagulation. Characterisation of animal models with Klkb1 deficiency and PK inhibition have demonstrated effects on inflammation, vascular function, blood pressure regulation, thrombosis, haemostasis, and metabolism. These reports have also identified a host of PK substrates and interactions, which suggest an expanded physiological role for this protease beyond the bradykinin system and coagulation. The review summarises the mechanisms that contribute to PK activation and its emerging role in diabetes and metabolism.

Topics: Adipogenesis; Animals; Blood Coagulation; Blood Pressure; Bradykinin; Diabetes Mellitus; Factor XII; Gene Expression Regulation; Genetic Variation; Glucose; Hemostasis; Humans; Hypertension; Inflammation; Mice; Plasma Kallikrein; Prekallikrein; Thrombosis

Plasma prekallikrein is the liver-derived precursor of the trypsin-like serine protease plasma kallikrein (PK) and circulates in plasma bound to high molecular weight kininogen. The zymogen is converted to PK by activated factor XII. PK drives multiple proteolytic reaction cascades in the cardiovascular system such as the intrinsic pathway of coagulation, the kallikrein-kinin system, the fibrinolytic system, the renin-angiotensin system and the alternative complement pathway. Here, we review the biochemistry and cell biology of PK and focus on recent in vivo studies that have established important functions of the protease in procoagulant and proinflammatory disease states. Targeting PK offers novel strategies not previously appreciated to interfere with thrombosis and vascular inflammation in a broad variety of diseases.

Topics: Animals; Aprotinin; Blood Coagulation; Bradykinin; Cerebral Hemorrhage; Complement System Proteins; Cysteine; Diabetic Retinopathy; Disulfides; Factor XIIa; Fibrinolysis; Hemostasis; Humans; Inflammation; Kallikrein-Kinin System; Kallikreins; Kinins; Mice; Oligonucleotides, Antisense; Peptides; Plasma Kallikrein; Protein Structure, Tertiary; Proteolysis; Renin-Angiotensin System; Signal Transduction; Thrombosis; Trypsin

Recent advances in combinatorial protein engineering have made it possible to develop non-Ig protein scaffolds that can potentially substitute for most whole antibody-associated properties. These protein scaffolds display most of the binding properties associated with the variable domain of antibodies. In theory, many different natural human protein backbones are suitable to be used as recombinant templates for engineering ; in practice however, only a few have yielded the necessary properties to be translated into << druggable biologicals >>. Amongst these properties, potential broad specificities towards any kind of target, ease of production, small size, good tolerability and low immunogenicity are essential. Intellectual property is another key issue. In this review, a particular emphasis will be given to the most validated non-Ig scaffolds that have reached the clinical development phase.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibodies; Antineoplastic Agents; Biopolymers; Clinical Trials as Topic; Drug Design; Drug Screening Assays, Antitumor; Fibronectins; Humans; Inflammation; Mice; Mice, Nude; Models, Molecular; Neoplasms; Neovascularization, Pathologic; Peptide Fragments; Peptides; Protein Conformation; Protein Engineering; Protein Structure, Tertiary; Receptors, LDL; Staphylococcal Protein A; Structure-Activity Relationship

Particulate matter (PM) is a key component of air pollutants and is associated with mortality of cardiovascular and respiratory diseases. PM-induced tissue injury involves inflammation and coagulation. Plasma prekallikrein (pKal), along with coagulation factor XII (FXII) and high-molecular-weight kininogen (HK), form the plasma kallikrein-kinin system (KKS), a component of the innate immune response that generates proinflammatory products in response to injury. When the KKS proteins contact with activation surface such as negatively charged molecules, this system becomes activated. Activated kallikrein (Kal) activates FXII to initiate the intrinsic coagulation pathway, and cleaves HK to release bradykinin to enhance vascular permeability and systemic inflammation. In his study we determined the role of plasma pKal in the PM

Topics: Animals; Blood Coagulation; Gene Deletion; Humans; Inflammation; Lung Injury; Mice; Mice, Knockout; Particulate Matter; Plasma Kallikrein

Essentials Zymogen PK is activated to PKa and cleaves substrates kininogen and FXII contributing to bradykinin generation. Monomeric PKa and dimeric homologue FXI utilize the N-terminal apple domains to recruit substrates. A high-resolution 1.3 Å structure of full-length PKa reveals an active conformation of the protease and apple domains. The PKa protease and four-apple domain disc organization is 180° rotated compared to FXI. SUMMARY: Background Plasma prekallikrein (PK) and factor XI (FXI) are apple domain-containing serine proteases that when activated to PKa and FXIa cleave substrates kininogen, factor XII, and factor IX, respectively, directing plasma coagulation, bradykinin release, inflammation, and thrombosis pathways. Objective To investigate the three-dimensional structure of full-length PKa and perform a comparison with FXI. Methods A series of recombinant full-length PKa and FXI constructs and variants were developed and the crystal structures determined. Results and conclusions A 1.3 Å structure of full-length PKa reveals the protease domain positioned above a disc-shaped assemblage of four apple domains in an active conformation. A comparison with the homologous FXI structure reveals the intramolecular disulfide and structural differences in the apple 4 domain that prevents dimer formation in PK as opposed to FXI. Two latchlike loops (LL1 and LL2) extend from the PKa protease domain to form interactions with the apple 1 and apple 3 domains, respectively. A major unexpected difference in the PKa structure compared to FXI is the 180° disc rotation of the apple domains relative to the protease domain. This results in a switched configuration of the latch loops such that LL2 interacts and buries portions of the apple 3 domain in the FXI zymogen whereas in PKa LL2 interacts with the apple 1 domain. Hydrogen-deuterium exchange mass spectrometry on plasma purified human PK and PKa determined that regions of the apple 3 domain have increased surface exposure in PKa compared to the zymogen PK, suggesting conformational change upon activation.

Topics: Binding Sites; Bradykinin; Factor XI; Humans; Inflammation; Kininogens; Mutation; Plasma Kallikrein; Prekallikrein; Protein Binding; Protein Domains; Protein Multimerization; Recombinant Proteins; Thrombosis

Recent evidence suggests that ischemic stroke is a thromboinflammatory disease. Plasma kallikrein (PK) cleaves high-molecular-weight kininogen to release bradykinin (BK) and is a key constituent of the proinflammatory contact-kinin system. In addition, PK can activate coagulation factor XII, the origin of the intrinsic coagulation cascade. Thus, PK triggers 2 important pathological pathways of stroke formation, thrombosis and inflammation.. We investigated the consequences of PK inhibition in transient and permanent models of ischemic stroke.. PK-deficient mice of either sex challenged with transient middle cerebral artery occlusion developed significantly smaller brain infarctions and less severe neurological deficits compared with controls without an increase in infarct-associated hemorrhage. This protective effect was preserved at later stages of infarctions as well as after permanent stroke. Reduced intracerebral thrombosis and improved cerebral blood flow could be identified as underlying mechanisms. Moreover, blood-brain barrier function was maintained in mice lacking PK, and the local inflammatory response was reduced. PK-deficient mice reconstituted with PK or BK again developed brain infarctions similar to wild-type mice. Important from a translational perspective, inhibition of PK in wild-type mice using a PK-specific antibody was likewise effective even when performed in a therapeutic setting up to 3 hours poststroke.. PK drives thrombus formation and inflammation via activation of the intrinsic coagulation cascade and the release of BK but appears to be dispensable for hemostasis. Hence, PK inhibition may offer a safe strategy to combat thromboembolic disorders including ischemic stroke.

Topics: Animals; Brain Infarction; Female; Inflammation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Plasma Kallikrein; Stroke; Thrombosis

Topics: Animals; Biomarkers, Tumor; Blood Pressure; Cardiovascular Diseases; Diabetes Mellitus; Female; Gene Expression Regulation, Neoplastic; Humans; Inflammation; Male; Mice; Neoplasms; Peptide Hydrolases; Plasma Kallikrein; Risk; Thrombosis; Tissue Kallikreins

Kallikrein acts on high molecular weight kininogen (HK) to generate HKa (cleaved HK) and bradykinin (BK). BK exerts its effects by binding to B(2) receptors. The activation of B(2) receptors leads to the formation of tissue plasminogen activator, nitric oxide (NO) and prostacyclin (PGI(2) ). An elevated kallikrein-dependent pathway has been linked to cardiovascular disease risk. The aim of this study was to investigate whether our novel plasma kallikrein inhibitor abolishes kallikrein-mediated generation of BK from HK and subsequent BK-induced NO and PGI(2) formation, thereby influencing endothelial pathophysiology during chronic inflammatory diseases.. Kinetic analysis was initially used to determine the potency of PF-04886847. Biochemical ligand binding assays, immunological methods and calcium flux studies were used to determine the selectivity of the kallikrein inhibitor. In addition, the effect of PF-04886847 on BK-induced relaxation of the rat aortic ring was determined in a model of lipopolysaccharide-induced tissue inflammation.. Evidence was obtained in vitro and in situ, indicating that PF-04886847 is a potent and specific inhibitor of plasma kallikrein. PF-04886847 efficiently blocked calcium influx as well as NO and PGI(2) formation mediated through the BK-stimulated B(2) receptor signalling pathway. PF-04886847 blocked kallikrein-induced endothelial-dependent relaxation of isolated rat aortic rings pre-contracted with phenylephrine.. PF-04886847 was shown to be the most potent small molecule inhibitor of plasma kallikrein yet described; it inhibited kallikrein in isolated aortic rings and cultured endothelial cells. Overall, our results indicate that PF-04886847 would be useful for the treatment of kallikrein-mediated inflammatory disorders.

Topics: Aminobenzoates; Aminopyridines; Animals; Bradykinin; Cells, Cultured; Endothelial Cells; Enzyme Inhibitors; Epoprostenol; Humans; Inflammation; Kinetics; Kininogen, High-Molecular-Weight; Lipopolysaccharides; Muscle Contraction; Nitric Oxide; Phenylephrine; Plasma Kallikrein; Rats; Signal Transduction; Substrate Specificity

Human plasma kallikrein (huPK) is a serine proteinase involved in many biological processes including those of the kallikrein-kinin system. The action of huPK on kininogen results in bradykinin (BK) release, a potent mediator of inflammatory responses. BK generation may be influenced by several agents, and the aim of this work was to investigate the effect of glycosaminoglycans (GAGs) on human high-molecular-weight kininogen (HK) hydrolysis by huPK and on inflammation. huPK was pre-incubated in the absence and presence of different GAGs, followed by the addition of kininogen. Bradykinin released at different times was measured by radioimmunoassay, and KM and kcat were calculated. Tuna and bovine dermatan sulfates, the most potent GAGs studied, reduced by 80% and 68%, respectively, the catalytic efficiency of huPK (control = 4. x 10(4) M(-1) s(-1) in BK release. The effect of bovine dermatan sulfate (BDS) on inflammatory response was studied in rat paw edema induced by carrageenin and hourly determined (1-4 h) by plethysmography. BDS significantly reduced the inflammatory response in the first and second hours of measurements (24% and 28%, respectively), p < 0.05. GAGs were shown to reduce bradykinin release "in vitro" and in an inflammation model. This reduction may play a role in the control or maintenance of some pathological and physiological processes.

Topics: Animals; Bradykinin; Carrageenan; Dermatan Sulfate; Edema; Humans; Hydrolysis; Inflammation; Inflammation Mediators; Kinetics; Kininogen, High-Molecular-Weight; Male; Plasma Kallikrein; Rats; Rats, Wistar